The present invention relates generally to a method of purifying plant proteins for use in nutritional products that have reduced levels of phytoestrogens, manganese and nucleic acids. More specifically, this invention is directed to a method of using ion exchange technology to remove phytoestrogens, manganese, nucleotides, nucleosides and RNA from plant proteins. This invention is also directed to the plant protein product resulting from the inventive process and to nutritional products that use the plant protein product as a source of amino nitrogen.
Phytoestrogens or plant estrogens occur in a variety of plants, including vegetable protein materials such as those derived from soybeans. Phytoestrogens are defined as plant substances that are structurally and functionally similar to the gonadal steroid 17 xcex2-estradiol or that produce estrogenic effects. There are three main groups of nonsteroidial dietary estrogens which are 1) the isoflavones, 2) the coumestans and 3) the mycoestrogens (fungal). The structural similarity between these substances and the endogenous mammalian estrogens have been studied. A review of phytoestrogens and their effects in mammals is reported by Kaldas and Hughes in an article entitled, xe2x80x9cReproductive and General Metabolic Effects of Phytoestrogens in Mammalsxe2x80x9d, Reproductive Toxicology, Vol. 3, pp. 81-89, 1989. The teachings of this article are herein incorporated by reference. As used in this specification and the appended claims, the term xe2x80x9cisoflavonesxe2x80x9d is equivalent to the term xe2x80x9cphytoestrogensxe2x80x9d as that term is defined in the Kaldas et al. article. Representative of the isoflavones that are reduced in plant proteins in accordance with the present invention are daidzein, daidzin, genistein and genistin.
Flavonoids and isoflavones are produced by numerous leguminosoe and grasses, including many plants commonly consumed by man and livestock. Soy isoflavones include compounds such as daidzin, genistin, daidzein and genistein. A general structural formula for these compounds is:
It has recently been recognized that isoflavones contained in vegetable proteins may have a detrimental impact upon the mammals that consume the vegetable protein, see Kaldas et al., supra. The concentration of isoflavones in plant protein isolates or concentrates such as soy protein isolates, can be as high as 3,000 xcexcg/g of protein. Isoflavones also provide the bitter or xe2x80x9cbeanyxe2x80x9d taste to vegetable proteins, (see Ewan et al. infra) may reduce the bioavailability of essential minerals and may influence the nutritional value of proteins (see Kaldas et al., supra). The consumption of isoflavones by man and livestock has also been connected with compromised reproductive systems in mammals. There is some concern that consumption of current soy based infant formulas that contain soy isoflavones may have an undesired physiological impact on the developing neuro-endocrine system of the infant. This concern is based in part, on evidence that soy-based animal feed may cause fertility problems in cheetahs. Setchell et al., 1987: xe2x80x9cGastroenterologyxe2x80x9d 93:225-33.
Further, the presence of high levels of manganese in body tissues has been suspected in the development of criminal behavior. See Gottschalk et al., xe2x80x9cAbnormalities in Hair Trace Elements as Indicators of Aberrant Behaviorxe2x80x9d, Compr Psychiatry 1991; 32:229-237, and Scientific American, March, 1995 pp. 104-105. Furthermore, there have also been reports that learning disabilities in children may be associated with increased levels of manganese in hair as reported by Collipp et al., in an article entitled, xe2x80x9cManganese in Infant Formula and Learning Disabilitiesxe2x80x9d, Ann. Nutritional Metals, 27:488-494, 1983. Typical plant protein isolates contain up to 1000 xcexcg of manganese per gram of protein. Thus, there is a need for improved processes that economically and on a commercial scale, provide for the reduction of isoflavone and manganese content in plant protein.
The use of nucleotides and nucleosides (or nucleotide equivalents as defined below) in nutritional formulas has received much attention in the last few years. It has been suggested that certain levels and ratios of the various nucleic acids can have a positive impact on the mammalian immune system and even prevent certain maladies such as diarrhea. The problem with using plant protein in such nutritional formulas is that the plant protein contains typically very high, inherent level of nucleic acids that may not be in the correct form (i.e., RNA) and at the correct ratios. Further, the high level of variation in the nucleic acid content causes problems in commercial manufacture. Typical plant protein isolates contain up to about 15 mg of nucleotide equivalents per gram of protein. Thus, the nutritional industry desires a source of plant protein that has substantially reduced levels of inherent nucleic acids. One additional benefit to the process of this invention is that, not only can the isoflavones and manganese be removed by the ion exchange column but also a substantial portion of the inherent nucleic acids.
Ion-exchange technology has been known for a great number of years. Ion-exchange resins are typically synthetic, insoluble, cross-linked polymers carrying acidic or basic side groups. They have high exchange capacities and can be used for an almost unlimited number of reactions. Ion-exchange resins are used in water-treatment, extraction, separation, analysis and catalysis.
Ion-exchange resins have an extended, open molecular framework that includes electrically charged ionic groups. A cation exchanger exchanges positive ions and therefore has negative ions built into its framework. An anion exchanger has positive ions in its framework. The ions of the lattice are called the fixed ions; the smaller ions of opposite charge that can change places with ions in the solution are called counterions.
Common problems encountered with ion exchange processes conducted on proteins include poor protein recovery (i.e., protein adhered to the resin) and inability of the protein slurry to pass through the resin bed resulting in a high pressure drop across the resin bed. The process which is disclosed herein fulfills the need in the nutritional industry for a source of plant protein that has highly reduced levels of isoflavones, manganese and nucleotides is economical, provides good protein recovery and can be used on a commercial scale.
U.S. Pat. No. 5,352,384 to Shen discloses a process to produce an isoflavone enriched vegetable protein fiber. This patent discloses the use of a glucosidase to convert the glucone isoflavones (i.e., daidzen) in a protein slurry to the aglucone isoflavones. The fiber fraction is then recovered from the slurry by centrifugation to provide an aglucone enriched fiber.
An article by Ewan et al. in the Journal of Food Science, Vol. 57, No. 2, 1992 entitled: xe2x80x9cIsoflavone Aglucones and Volatile Organic Compounds in Soybeans; Effects of Soaking Treatmentsxe2x80x9d, discloses the beneficial effects of soaking soybeans in mildly alkaline NaHCO3 solutions at elevated temperatures, for manufacturing soymilk with improved flavor. This publication does not suggest or disclose the use of an ion-exchange resin to remove isoflavones, manganese and nucleic acids from plant protein.
In an article published in volume 47 (1982) of the Journal of Food Science, pp. 933-940, by J. How and C. Morr entitled xe2x80x9cRemoval of Phenolic Compounds from Soy Protein Extracts Using Activated Carbonxe2x80x9d, they report subjecting soy protein extracts to activated carbon and ion exchange process treatments to remove phenolic compounds that have been reported as being responsible for adverse color and flavor characteristics of soy protein products. Protein extracts were subjected to a two stage, sequential ion exchange treatment prior to protein precipitation. The protein extract was pumped xe2x80x9cdown-flowxe2x80x9d through a cation exchange column in the Na+ form and then an anion exchanger in the hydroxyl and chloride form to remove polyvalent anions including phenolic acids, phytate and others.
U.S. Pat. No. 5,248,804 to Nardelli et al. discloses a process for the removal of phytate from plant protein using ion-exchange resins. The process uses a macroporous anion exchange resin (weak base or strong base) which has been conditioned by 1) conversion to the hydroxide form; 2) conversion to the chloride or sulfate form; and 3) thereafter conversion of the strong base sites to the carbonate form and the weak base sites to the free base form. The plant protein containing phytate is then contacted with the treated resin to remove the phytate. The teachings of U.S. Pat. No. 5,248,804 are herein incorporated by reference.
Phytate comprises the salts of phytic acid. Phytic acid is also known as inositol hexaphosphate. Thus, in using an anion exchange resin, the highly anionic phosphate groups provide the handle by which the resin can extract the phytate from the protein slurry. In contrast, isoflavones and nucleotides are neutral molecules and would not be expected to attach to the resin or exchange with the anions on the resin.
U.S. Pat. No. 5,492,899 to Masor et al. discloses an infant formula with ribo-nucleotides. This patent teaches the use of certain levels and ratios of nucleotide equivalents in infant formulas and discloses an analytical technique to identify and quantify the nucleotide equivalents in a nutritional matrix. As used herein and in the claims of this invention, the term xe2x80x9cnucleotidexe2x80x9d is the same as the term xe2x80x9cnucleotide equivalentxe2x80x9d as defined in U.S. Pat. No. 5,492,899. U.S. Pat. No. 5,492,899 defines nucleotide equivalents as polymeric RNA, ribo nucleosides, ribo-nucleosides containing adducts and mono-, di- and triphosphate ribonucleotides. The teachings of U.S. Pat. No. 5,392,899 are herein incorporated by reference.
The present invention comprises a method through which low isoflavone, low manganese or low nucleotide plant proteins can be manufactured. The invention further comprises the low isoflavone, low manganese and low nucleotides protein isolates themselves and to such protein isolates that are produced according to the method of the present invention. The present invention further comprises nutritional products made with the protein isolates produced in accordance with the invention. This, and other aspects of the invention are specifically described in detail in the description set forth below.
In its broadest application, the present invention relates to a method of reducing the isoflavone, manganese or nucleotide content of a plant protein comprising:
a) providing at least one anion exchange resin;
b) providing a slurry of plant protein that contains isoflavones, manganese or nucleotides;
c) contacting said slurry with said anion exchange resin; and
d) separating said slurry with reduced content of isoflavones, manganese or nucleotides from said anion exchange resin.
Representative counterions useful in the anion exchange resin according to this invention, include acetate, citrate, chloride, bisulfate, carbonate and bicarbonate. As most anion exchange resins are supplied in the chloride form, it is possible to use such chloride resins directly without pretreatment. As discussed below, a preferred procedure for resin pretreatment washes the chloride resin with caustic to clean the resin, then a HCl wash is conducted to clean and control microbial growth and then the resin is converted to the carbonate and/or bicarbonate form.
In the production of plant protein using the process according to this invention, the anion that is released from the resin as a result of entrapping the isoflavone, manganese or nucleotide is important to the quality of the finished product. That is to say, the resulting protein should not be denatured, contain unacceptable levels of free hydroxyl groups or other offensive anions (i.e., chloride) that would produce a protein product that would be unacceptable for use in a nutritional product. For example, typical soy protein isolate contains sufficient levels of isoflavones, manganese and nucleotides that treatment with an anion exchange resin that has chloride as the counterion would produce a resulting protein that has excessive levels of chloride. In similar fashion, if the counterion is hydroxyl, the resulting product would need to be treated with acid to neutralize the basic product, thus unacceptably increasing the mineral load associated with the protein.
In one preferred embodiment of this invention, the anion exchange resin uses a counterion, such as carbonate or bicarbonate, which avoids the aforementioned problems. As used in the specification and in the appended claims, the term xe2x80x9ccarbonatexe2x80x9d means carbonate and bicarbonate.
There is disclosed a method of reducing the isoflavone, manganese or nucleotide content of plant protein comprising:
a) providing at least one anion exchange resin containing strong base sites and weak base sites, said anion exchange resin prior to step b), being subjected to the steps comprising:
i) conversion to a hydroxide form;
ii) conversion to a chloride or sulfite form; and
iii) conversion of at least some of said strong base sites to the carbonate form and at least some of said weak base sites to the free base form;
b) providing a slurry of plant protein;
c) contacting said slurry with said anion exchange resin; and
d) separating said slurry with reduced content of isoflavones, manganese or nucleotides from said anion exchange resin.
There is further disclosed a method for separating compounds selected from isoflavone, manganese or nucleotides from a plant protein slurry, said method comprising the steps of:
a) selecting an anion exchange resin;
b) exposing the resin to an agent that places on the resin an exchangeable anion that:
i) does not change the pH of the protein slurry outside the range of 6.0 to 9.5; and
ii) does not add an objectionable anion to the effluent protein slurry at step d);
c) providing a slurry containing a source of plant protein and at least one compound selected from isoflavone, manganese and nucleotides;
d) bringing the resin into contact with the resin; and
e) separating the slurry from the resin.
The present invention also relates to the protein that results from the process described herein. The protein product or isolate according to this invention is characterized in that it contains less than 30 xcexcg of isoflavones per g of protein, less than 450 xcexcg of manganese per g of protein and less than 10 mg of nucleotides per g of protein. The present invention further relates to a plant protein composition which comprises less than 30 xcexcg of isoflavones per g of plant protein and to nutritional products comprising said protein.
There is also disclosed infant formulas that are based on plant protein and contain less than 600 xcexcg of isoflavones per liter of ready-to-feed formula, more preferably less than 200 xcexcg and most preferably less than 100 xcexcg.
Typically, the method of this invention is conducted by placing the anion exchange resin in a bed, column or reactor through which the protein slurry is passed. The bed, column or reactor has at least one inlet and at least one outlet and is preferably operated as a vertical column in the xe2x80x9cupflowxe2x80x9d mode. In another embodiment, the preconditioned resin may be added to a tank containing the protein slurry and after an appropriate period of time for the reaction to take place, the resin is filtered from the slurry.
The anion exchange resin is typically a macroporous resin and is preferably a Type I or II macroporous resin. In a preferred embodiment, the anion exchange resin is selected from weak base anion exchange resins, strong base anion exchange resins and mixtures thereof. Representative of the anion exchange resins useful in the present invention include Amberlite(copyright) RA95, IRA-910 and IRA-900 sold by Rohm and Haas Company, Dowex-22 and MSA-1 sold by Dow Chemical and Purolite A510 and A500 sold by Purolite Company. As used herein and in the claims, the term resin is meant to include gels, which those skilled in the art would understand to be useful in the process described herein. Representative of such gels are Amberlite(copyright) IRA 410 (Type II gel, strong base anion) sold by Rohm and Haas Company, IRA 402 is a Type I strong base anion exchange gel that is not macroporous that would also be useful.
The proteins that may be used in the method of this invention include any plant protein that contains detectable levels of isoflavones, manganese and nucleotides. More specifically, the protein is obtainable from soybeans, corn, wheat, peas, beans, cottonseed, peanuts, carrots, alfalfa, apples, barley, bluegrass, clovers, coffee, garlic, hops, marijuana, oats, algae, orchard grass, parsley, rice, rye, sage, sesame, yeast, fungus, potatoes, hydrolyzates thereof and mixtures thereof.
It is preferred that the protein be a protein isolate or concentrate wherein the levels of fats, oils and carbohydrates have been reduced. It has been determined that the presence of fats and oils reduces the efficiency of the inventive process.
The chemical agents useful in converting the resin to the hydroxide form include sodium hydroxide, calcium hydroxide, potassium hydroxide and magnesium hydroxide. The most preferred agent is sodium hydroxide.
The chemical agents useful in converting the resin to the chloride or sulfate form include hydrochloric acid, sulfuric acid and sodium chloride. The preferred agent is hydrochloric acid.
The chemical agents useful in converting the resin to the carbonate or free base form include any of the weak base salts such as sodium carbonate, sodium bicarbonate and ammonium hydroxide. Sodium bicarbonate is the most preferred agent as it produces a protein effluent at a pH range of 6.6-9.5.
Those skilled in the art of ion exchange technology will appreciate that the protein slurry containing the isoflavones, manganese or nucleotides, as it is contacted with the anion exchange resin, should be at a pH that does not denature the protein, which causes clogging of the column. Further, adjustment of the pH past neutral, will add significant levels of anions to the slurry which will compete for counterion sites. Typically, a pH of from about 5.5 to 10 is satisfactory. Preferably, the pH of the protein slurry feed can range from 6.0 to 8.0. The pH of the protein slurry effluent (leaving the column or bed) should be near the pH at which the protein will be used in a final product. Thus, if a plant protein treated in accordance with this invention is to be used in an infant formula, the effluent pH should be about 6.0 to 7.5. In a preferred embodiment, the plant protein feed to the resin should be as free of added anions (i.e., xe2x80x94OH, Clxe2x80x94, and the like) as possible. The addition of acids, bases, salts and the like to the protein slurry feed decreases the efficiency of the column to remove the isoflavones, manganese or nucleotides from the protein slurry.
As those skilled in the art will appreciate, exchange resins have a finite capacity and may be regenerated to an active state after exhaustion or near exhaustion. Thus, as contemplated in this invention, the exchange resins after contact with the plant protein is regenerated or reconditioned through known steps to the anionic form or more preferably through the steps comprising:
1) stripping the resin of any residue (i.e., protein) and conversion to the hydroxide form, for example through the use of sodium hydroxide;
2) conversion of the resin to the chloride or sulfate form; and
3) conversion of the strong base sites on the resin to the carbonate form and conversion of the weak base sites to the free base form.
Those skilled in the ion exchange resin art will appreciate that non-aqueous and alcohol water regenerations can be used.
One preferred embodiment of the method according to the present invention includes the step of homogenizing the plant protein slurry prior to contact with the resin. Homogenization or treatments similar thereto have been found in the process of this invention, to increase the effective removal of isoflavones, manganese and nucleotides from the slurry. In addition, homogenization of the protein slurry prior to contact with the resin reduces the pressure drop across the resin bed or column which facilitates the facile and economic production of a plant protein for use in nutritional products.
The present invention is also directed to a plant protein isolate that has specified levels of isoflavones and to a plant protein that has been subjected to the process disclosed herein and to nutritional products that are made from such proteins. Also contemplated herein are animal feeds that are substantially free of isoflavones. More specifically, the present invention relates to a plant protein containing less than about 30 xcexcg of isoflavones per g of protein, less than about 450 xcexcg of manganese and less than about 10 mg of nucleotide equivalents per gram of protein. In a more preferred embodiment, the protein is derived from soy beans and contains less than 20 xcexcg isoflavone per gm of protein. In a most preferred embodiment, the plant protein contains less than 10 xcexcg isoflavone per gin of protein, less than 5 mg of nucleotides per gm of protein and less than 200 xcexcg of manganese per g of protein.
The following Examples describe specific, but non-limiting, embodiments of the present invention. The features of the present invention which are believed to be novel are set forth with particularity in the appended claims and should be understood as to structure and manner of operation by the following detailed Examples.